The ultimate objective of Project 3 is aimed at the discovery of potent and specific synthetic organic compounds that either activate or inhibit human PAS kinase (PASK). Three interdependent projects will be simultaneously undertaken in pursuit of this objective. The first project will entail genetic, biochemical and molecular biological studies aimed at further defining the biological role of the PASK enzyme. Although significant progress regarding the biology of PASK has already been made, the proposed experimental plan seeks to resolve the identities of interacting genes and enzymatic substrates. Such efforts promise to deliver a concise and accurate description of the regulatory pathway controlled by PASK. They may further lead to an understanding of the manner in which the two PAS domains sense physiological signals and thereby regulate the activity of the catalytic, serine/threonine kinase domain of the enzyme. The second project will use high throughput drug screening (HTS) as a means of identifying small, synthetic chemicals capable of either activating or inhibiting PASK. The intact enzyme will be over-expressed in baculovirus-infected insect cells, purified and tested in HTS assays using a library of 350,000 distinct, druglike chemicals. The third project will employ NMR spectroscopy as a means of resolving the structures of the two PAS domains and identifying synthetic chemicals capable of avid interaction with the native enzyme. Progress to date has led to a high resolution structure of the PAS-A domain as well as the identification of synthetic chemicals that bind to this domain in a specific and logical manner. The proposed efforts to solve the NMR structure of the PAS-B domain and undertake comprehensive NMR-based screening constitute a rational approach that is well balanced with respect to the HTS biochemical assay for synthetic modulators of the PASK enzyme. The integrated execution of all three projects offers a favorable opportunity for the discovery of drug-like molecules capable of modulating the mitotic growth of mammalian cells.